Cinnamaldehyde derivative compounds, and methods of use for cinnamaldehyde derivative compounds nicotine cessation

ABSTRACT

Compounds, compositions, and methods of use of such compounds and compositions are provided for reducing human dependency to nicotine. In one example, a method of treating an individual with an addiction to nicotine comprises administering to the individual a compound that is a structural analog of trans-cinnamaldehyde ((2E)-3-Phenylprop-2-enal):Based on the administration, a rate at which nicotine is metabolized may be reduced, which in turn may reduce a desire for the individual to consume nicotine-containing products.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application claims priority to U.S. Provisional ApplicationNo. 62/750,147, entitled “AGENTS FOR NICOTINE CESSATION ANDCHEMOPREVENTION,” and filed on Oct. 24, 2018. The entire contents of theabove-referenced application are hereby incorporated by reference forall purposes.

ACKNOWLEDGMENT OF GOVERNMENT SUPPORT

This invention was made with government support under Grant Number:1R15DA042341-01 awarded by the National Institute on Drug Abuse. Thegovernment has certain rights in this invention.

FIELD OF THE INVENTION

The present invention pertains to compounds, compositions, and methodsof use for chemical compounds found to inhibit the primary humannicotine metabolizing enzyme, cytochrome P450 2A6 (CYP2A6). Thecompositions and methods of use may reduce a frequency at which productscontaining nicotine are consumed, and may reduce an exposure ofindividuals to carcinogenic substances over time.

BACKGROUND OF THE INVENTION

Nicotine is the addictive agent in cigarettes. Tobacco users alter theirsmoking behavior/smoke exposure to maintain nicotine plasmaconcentrations within a defined range (e.g., 10 to 50 ng/mL). Nicotineelimination occurs mainly through metabolism, and factors reducingnicotine metabolism may result in fewer cigarettes smoked per day due tonicotine concentrations being maintained for longer periods. Conversely,rapid nicotine metabolism may result in elevated smoking rates andcarcinogen exposure, as smokers must increase nicotine exposure tomaintain plasma concentrations. Although nicotine addiction is a complexdisease involving many factors, nicotine plasma concentration is acentral factor.

Tobacco addiction is the leading global cause of preventable death.Aside from contributing to preventable death, tobacco addiction posesother substantial problems, such as diverting funds from other importanthealth problems, and disproportionately impacting lower incomefamilies/individuals. Considering the adverse impact of tobaccoaddiction on health, the current number of treatments for decreasingtobacco use is severely deficient. Unlike many disease states for whichthere are many treatment options to account for variability in patientresponse, currently there are only three pharmacological approachesapproved by the U.S. Food and Drug Administration to treat nicotineaddiction. These include nicotine replacement therapy, bupropion, andvarenicline. Alternative approaches (e.g. meditation, hypnosis, andothers) have been used but the effectiveness of these approaches isvague or has not been studied extensively. By contrast, there are 29drugs for depression and 33 for diabetes, for example. Thus, there is aneed for compounds that can reduce the urge to smoke (or consume othernicotine-containing products), thereby reducing a potential for adversehealth implications.

Cytochrome P450 2A6 (CYP2A6) is an enzyme responsible for metabolizing70-80% of a nicotine dose. CYP2A6 displays wide genetic variability withmore than 37 known polymorphisms to date. Some of these polymorphismsresult in a CYP2A6 enzyme with reduced nicotine metabolizing activity.Genetic-based differences in nicotine metabolism influence smokingbehavior and cigarette smoke exposure. Indeed, individuals with CYP2A6variants that are poor metabolizers of nicotine smoke fewer cigarettesand have higher smoking cessation success rates than individualsexpressing a variant of the CYP2A6 enzyme that extensively metabolizesnicotine.

It has been previously shown that trans-cinnamaldehyde (t-CA), alsoreferred to herein as CA), comprising a low-molecular weightphenylpropanoid present in cinnamon (which contributes to cinnamon'sflavor and aroma and is a major component of cinnamon oil), is amechanism-based irreversible inhibitor (MBI) of CYP2A6. MBIs are alsooften referred to as time-dependent inhibitors (TDI), particularly inthe drug-drug interaction literature associated with the pharmaceuticalindustry and related guidance from the United States Food and DrugAdministration.

It is herein recognized that genetic variability (>37 polymorphisms todate, as discussed above) with regard to CYP2A6 may result in t-CA beingeffective in inhibiting CYP2A6 in some cases (thereby slowing a rate atwhich nicotine is metabolized), while not being as effective in othercases. Thus, it is herein recognized that there may be particularstructural analogs of t-CA which may be more effective in certain cases,as compared to others. Furthermore, it is herein recognized that theremay be structural analogs of t-CA which are more potent than t-CA, andwhich may thus be more effective at reducing an urge to consumenicotine-containing products.

Another cytochrome p450 enzyme CYP2A13, is localized in the lung, andshows overlapping substrate specificity with CYP2A6. Specifically, ithas been previously shown that CYP2A13 can catalyze the metabolism ofnicotine. CYP2A13 also catalyzes the metabolism of tobacco-specificnitrosamines, which contributes to carcinogenic processes and lungcancer. Accordingly, it is herein recognized that particular structuralanalogs of t-CA may in some examples inhibit CYP2A13, which may reduce arate at which nicotine is metabolized and also reduce the activation oftobacco-specific carcinogens to metabolites that alkylate DNA.

SUMMARY

The present disclosure provides compounds, compositions, and methods ofuse for such compounds and compositions that may possess improvedability to inhibit/inactivate CYP2A6. The improved ability may relate toan increased inhibition potency as compared to t-CA, for example. Theimproved ability may relate to a particular t-CA structural analogdisplaying increased potency for a particular CYP2A6 genotype, in otherexamples. Similarly, the improved ability may relate to a particulart-CA structural analog being more effective than other t-CA structuralanalogs (or t-CA itself) with regard to how nicotine is metabolized in aparticular individual (e.g., whether the particular individual is anultrafast, fast, intermediate, or slow metabolizer of nicotine). In someexamples, the compounds, compositions and methods of use for suchcompounds and compositions may additionally or alternatively possess anability to inhibit/inactivate CYP2A13.

The compounds of the present disclosure may includeα-bromocinnamaldehyde, α-chlorocinnamaldehyde, 2-nitrocinnamaldehyde,2-trifluoromethylcinnamaldehyde, 2,6-difluorocinnamaldehyde,2-methoxycinnamaldehyde, 3-nitrocinnamaldehyde, 4-nitrocinnamaldehydeand 2-nitro-α-methylcinnamaldehyde, 2-nitro-α-ethylcinnamaldehyde,2-nitro-α-propylcinnamaldehyde, 2-nitro-α-isopropylcinnamaldehyde,2-nitro-α-butylcinnamaldehyde, 2-nitro-α-amylcinnamaldehyde,2-nitro-α-hexylcinnamaldehyde, and hydrocinnamaldehyde. Theabove-mentioned compounds may be time-dependent inhibitors of CYP2A6and/or CYP2A13. In some examples, a method for treating an individualwith an addiction to nicotine may comprise administering to theindividual one or more of the above-mentioned chemical structures,wherein a rate at which nicotine is metabolized is reduced uponadministration of the compound to the individual. In such a method, agenetic variant of CYP2A6 may in some examples be determined, whereinselection of the compound to administer may be based on the geneticvariant. In some examples, selection of the compound to administer maybe based on whether the individual is an ultrafast, fast, intermediate,or slow metabolizer of nicotine. The one or more compounds may beadministered orally to the individual.

Another embodiment of the present disclosure may include apharmaceutical composition for reducing a rate at which nicotine ismetabolized, where the pharmaceutical composition comprises one or moreof the above-mentioned compounds (α-bromocinnamaldehyde,α-chlorocinnamaldehyde, 2-nitrocinnamaldehyde,2-trifluoromethylcinnamaldehyde, 2,6-difluorocinnamaldehyde,2-methoxycinnamaldehyde, 3-nitrocinnamaldehyde, 4-nitrocinnamaldehydeand 2-nitro-α-methylcinnamaldehyde, 2-nitro-α-ethylcinnamaldehyde,2-nitro-α-propylcinnamaldehyde, 2-nitro-α-isopropylcinnamaldehyde,2-nitro-α-butylcinnamaldehyde, 2-nitro-α-amylcinnamaldehyde,2-nitro-α-hexylcinnamaldehyde, 2-methoxycinnamaldehyde, andhydrocinnamaldehyde). For such a composition, the rate at which nicotineis metabolized may be reduced based on the pharmaceutical compositioninhibiting an enzyme (e.g., CYP2A6, CYP2A13) that metabolizes nicotine.The composition may in some examples further comprise an oil.Additionally or alternatively, the composition may further comprise asurfactant. Additionally or alternatively, the composition may furthercomprise a co-surfactant.

Another embodiment of the present disclosure includes structural analogsof t-CA (such as the compounds discussed above), additionally designedto specifically protect t-CA (or structural analogs thereof) from beingdegraded. Specifically, while t-CA is well-absorbed in the intestinaltract following oral administration, clearance is rapid, as the aldehydegroup of t-CA (and structural analogs thereof) may be susceptible tometabolism by a variety of pathways. Thus, in one aspect of the presentinvention, the rate of t-CA (or structural analogs thereof) degradationmay be reduced by utilizing a ‘prodrug’ oxime-ether form of t-CA (orstructural analogs thereof), which may protect the aldehyde group fromdegrading as the t-CA (or structural analogs thereof) travels to itstherapeutic target.

In another aspect of the disclosure, the rate of degradation of t-CA (orstructural analogs thereof) may be reduced by, as a first example,utilizing a first formulation that includes a co-solvent solution usingtri-block co-polymers. t-CA (and structural analogs thereof) may beincorporated into such tri-block co-polymers by a solvation method. Inone example, the tri-block co-polymers (e.g., polaxamers) are selectedbased on an enhancement in the oral bioavailability of t-CA (andstructural analogs thereof). However, other tri-block co-polymers may beused without departing from the scope of this disclosure. In someexamples, block co-polymers comprising PEG-PCL or PEG-PLA, may be usedto obtain higher drug concentrations.

In another aspect of the disclosure, a self-emulsifying drug deliverysystem (SEDDS) may be utilized to formulate a second formulation thatincludes t-CA or structural analogs thereof. Briefly, a SEDDS maycomprise physically stable mixtures of oil, surfactants, co-surfactantsand solubilized drug substances (e.g. t-CA and structural analogsthereof), which may then be administered orally (in soft or hard gelatincapsules for example). The oil may be a long chain triglyceride, amedium chain triglyceride, etc., with varying degrees of saturation. Thesurfactants may be anionic surfactants, cationic surfactants, ampholyticsurfactants, nonionic surfactants, etc. Examples of anionic surfactantsof the present disclosure include potassium laurate and sodium laurylsulphate. An example of a cationic surfactant of the present disclosureincludes quaternary ammonium halide. An example of ampholyticsurfactants of the present disclosure include sulfobetaines. An exampleof nonionic surfactants of the present disclosure include sorbitanesters (Spans) and polysorbates (Tweens). In a gastrointestinal tractenvironment, the SEDDS may spontaneously emulsify. In developing theSEDDS, particular surfactant/particular mixtures of surfactants may beselected to match a Hydrophile-Lipophile Balance (HLB) value for t-CAoil (or structural analogs thereof). t-CA (and structural analogsthereof) loading and stability may be assessed by monitoring size andzeta potential. t-CA (and structural analogs thereof) release may beassessed by a dialysis method under sink conditions.

In this way, an individual's dependence on nicotine may be reduced. Insome examples, by relying on the compounds, compositions, formulationsand methods discussed herein, an individual may be able to completelyquit their use of nicotine-containing products. Dosages and selection ofparticular compounds may in some examples be based on how an individualmetabolizes nicotine. For example, a first compound may be administeredto an individual who is determined to be a fast metabolizer of nicotine,whereas a second compound may be administered to an individual who isdetermined to be a slow metabolizer of nicotine. For example, the firstcompound may have a greater potency with regard to CYP2A6 inhibition, ascompared to the second compound. In other examples, a same compound maybe administered to both an individual that is a fast metabolizer ofnicotine and an individual that is a slow metabolizer of nicotine, but adosage may be greater for the individual that is the fast metabolizer,as compared to the individual that is the slow metabolizer. Determiningwhether an individual is a fast or slow (or in other examples alsoultrafast and intermediate) metabolizer of nicotine may be accomplishedby methodology readily available to those of ordinary skill in the art.As examples, nicotine metabolism in an individual may be measured (e.g.,by monitoring nicotine metabolites as will be elaborated in furtherdetail below), may be determined based on a genetic profile of CYP2A6(or in some examples CYP2A13) for the individual, or may bequalitatively determined based on an interview or questionnaire providedto the individual who desires to reduce their dependence on nicotine.

It should be understood that the summary above is provided to introducein simplified form a selection of concepts that are further described inthe detailed description. It is not meant to identify key or essentialfeatures of the claimed subject matter, the scope of which is defineduniquely by the claims that follow the detailed description.Furthermore, the claimed subject matter is not limited toimplementations that solve any disadvantages noted above or in any partof this disclosure.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts a scheme whereby nicotine is metabolized to cotinine byCYP2A6;

FIG. 2 depicts an example illustration of different metabolism rates ofnicotine for individuals that are slow metabolizers of nicotine ascompared fast metabolizers of nicotine;

FIG. 3A depicts a structural representation of trans-cinnamaldehyde(t-CA);

FIG. 3B depicts a reaction scheme whereby t-CA inhibits CYP2A6;

FIGS. 4A-4C depict options for chemical substitutions to t-CA;

FIGS. 5A-5B depict a select number of structural analogs of t-CAprepared via substitutions as depicted at FIGS. 4A-4C;

FIG. 6 depicts an example dataset of dissociation constants forstructural analogs of CA as determined via a molecular docking approach;

FIG. 7A illustrates that potent inhibition of CYP2A6 requires both CAand NADPH and increases with time;

FIG. 7B depicts a representative data plot for obtaining k_(inact) andk_(I) values;

FIG. 8 depicts a number of structural analogs of t-CA of the presentdisclosure found to not display time-dependent inhibition of CYP2A6;

FIG. 9A depicts an example reaction scheme of routes whereby anoxime-ether is metabolized to t-CA (or structural analogs thereof);

FIG. 9B depicts examples of oxime ethers of the present disclosure,which may be used for structural analogs of t-CA;

FIG. 10 depicts a general synthetic scheme for oxime ethers of t-CA andstructural analogs of t-CA; and

FIG. 11 depicts a synthetic scheme for 2-nitro-alpha-alkylcinnamaldehydeanalogs of the present disclosure.

DETAILED DESCRIPTION OF THE INVENTION

The compounds, formulations, compositions and/or methods of use of suchcompounds and/or formulations discussed herein may improve an abilityfor individuals with an addiction to nicotine to reduce theirconsumption of nicotine-containing products, which may thus result insuch individuals reducing a potential risk for adverse healthimplication that result from their consumption of nicotine-containingproducts.

Turning to FIG. 1, example scheme 100 illustrates that CYP2A6metabolizes nicotine to an iminium ion, which is further metabolized viaaldehyde oxidase to cotinine, the major blood-circulating metabolite ofnicotine. As depicted, cotinine may be further metabolized by CYP2A6 tohydroxycotinine. As mentioned above, a rate at which nicotine ismetabolized via the example scheme 100 may be different for differentindividuals based on their particular CYP2A6 genetic subtype. As isknown in the art, it may be possible to determine rates of metabolism ofnicotine for different individuals by evaluating ahydroxycotinine/cotinine ratio as a function of time in blood samples ofdifferent individuals after consumption of nicotine-containing products.In another example, as is known in the art, it may be possible to inferrates of metabolism (e.g., ultrafast, fast, intermediate, slow) based ondetermined CYP2A6 genotype. Briefly, DNA or mRNA from a biologicalsample of an individual may be obtained, and the CYP2A6 subtype may bedetermined by amplifying the CYP2A6 region to prepare a DNA sampleenriched in DNA from the CYP2A6 gene region, and then sequencing the DNAsample to determine if the individual is an ultrafast, fast,intermediate, or slow metabolizer of nicotine. In other examples, suchmethodology may simply characterize an individual as a fast metabolizeror a slow metabolizer. By determining rates of metabolism of nicotine indifferent individuals, it may be possible to select a particularstructural analog of t-CA (see FIGS. 4A-5B) that are more effective fora particular individual. In other words, a particular t-CA structuralanalog may be more effective than another t-CA structural analog for anindividual determined to be a fast metabolizer of nicotine, for example.

For illustrative purposes, FIG. 2 depicts example illustration 200,illustrating a relationship between nicotine concentration in blood(y-axis), over time (x-axis), for an individual with a CYP2A6 variantthat rapidly metabolizes nicotine (line 205), compared to an individualthat slowly metabolizes nicotine (line 210). Solid arrows 206 correspondto peaks of line 205, and represent times when a cigarette is smoked forthe individual that rapidly metabolizes nicotine. Similarly, dashedarrows 211 correspond to peaks of line 210, and represent times when acigarette is smoked for the individual that slowly metabolizes nicotine.As can be seen, the individual that metabolizes nicotine slower smokesless over time (three cigarettes as compared to five). The compounds(e.g., structural analogs of t-CA) discussed herein may, for example,modify an individual's nicotine metabolism such that when the compoundis administered, the individual has a reduced desire to consumenicotine-containing products.

Specifically, based on the relationship between nicotine metabolism andsmoking behavior, as smokers typically ‘wean’ off nicotine and continueto smoke while they attempt to quit, CYP2A6 inhibitors may help smokersmaintain blood nicotine concentrations for longer periods, such that atime between smoking episodes may be extended, which may thus improveefforts to resist the routines (e.g., “smoke breaks”) that contribute tosmoking addiction and potential adverse health consequences stemmingtherefrom.

Turning to FIG. 3A, depicted is a chemical structure 300 of t-CA. Asmentioned above, t-CA is an MBI (also referred to as a TDI) of CYP2A6.Accordingly, FIG. 3B depicts an illustrative scheme 350 for themechanism-based inhibition of CYP2A6. Specifically, t-CA 300 undergoesmetabolism by CYP2A6 (depicted as arrow 360), such that t-CA 300 ismetabolized to a CA reactive metabolite 351. In the presence of CYP2A6352, a step 365 in which the CA reactive metabolite 351 binds (depictedas line 356) CYP2A6 352 results in an inactivated CYP2A6 355. BecauseMBIs such as t-CA irreversibly inactivate the enzyme to which they bind,MBIs may offer an advantage in that they may provide a prolongedinhibition (prolonged nicotine inhibition in the case of CYP2A6inhibition), as compared to reversible inhibitors. In other words,because the particular enzyme (e.g. CYP2A6) becomes permanentlyinactivated, in order to reinstate the metabolizing activity of theparticular enzyme, the particular enzyme may first have to beregenerated, which may be dependent on the intrinsic rate of expressionof the particular enzyme in a particular cell type (e.g. intrinsic rateof expression of CYP2A6 in hepatocytes).

FIGS. 3A-3B depict t-CA for illustrative purposes. As discussed above,it is herein recognized that t-CA may effectively inhibit CYP2A6 forsome individuals, but may be less effective or ineffective in otherexamples depending on the individual's CYP2A6 genotype. It is furtherherein recognized that structural analogs of t-CA may provide greaterpotential for CYP2A6 inhibition than t-CA, which may further be afunction of CYP2A6 genotype (or in other words, based on how nicotine ismetabolized in particular individuals). Thus, there may be particularadvantages to the use of structural analogs of t-CA in terms of reducingconsumption of nicotine-containing products and thereby reducingparticular individual's exposure to carcinogenic substances.

The structural analogs of t-CA relevant to the present disclosure arenow discussed. As one example, FIG. 4A shows example illustration 400,depicting t-CA substituted at the alpha position. Structural analogs ofthe present disclosure as depicted at FIG. 4A may include a halogensubstituted at the alpha position of t-CA, for example. Specifically,FIG. 5A depicts a set of compounds 500 relevant to the presentdisclosure. Compounds (1) and (2) at FIG. 5A illustrate structuralanalogs substituted with a halogen at the alpha position of t-CA.Specifically, compound (1) is α-bromocinnamaldehyde, and compound (2) isα-chlorocinnamaldehyde.

FIG. 4B shows example illustration 425, depicting t-CA substituted atthe ring position. Structural analogs of the present disclosure asdepicted at FIG. 4B may include substitution at one or more of theortho, meta, and para positions of the ring. Examples of such compoundsrelevant to the present disclosure are depicted at FIG. 5A.Specifically, ortho-substituted analogs of t-CA relevant to the presentdisclosure are depicted by compounds (3), (4), (5), and (6). Compound(3) is 2-nitrocinnamaldehyde, compound (4) is2-trifluoromethylcinnamaldehyde, compound (5) is2,6-difluorocinnamaldehyde, and compound (6) is 2-methoxycinnamaldehyde.A meta-substituted analog of t-CA relevant to the present disclosure isdepicted by compound (7). Compound (7) is 3-nitrocinnamaldehyde. Apara-substituted analog of t-CA relevant to the present disclosure isdepicted by compound (8). Compound (8) is 4-nitrocinnamaldehyde.

FIG. 4C shows example illustration 450, depicting t-CA substituted atboth the alpha position and the ring position. An example of such acompound relevant to the present disclosure is depicted by compound (9)at FIG. 5A. Compound (9) is 2-nitro-α-methylcinnamaldehyde. Otherexamples of structural analogs of t-CA substituted at both the alphaposition and the ring position are depicted by compounds (10)-(15) atFIG. 5B. Specifically, compound (10) is 2-nitro-α-ethylcinnamaldehyde,compound (11) is a 2-nitro-α-isopropylcinnamaldehyde, compound (12) is2-nitro-α-propylcinnamaldehyde, compound (13) is2-nitro-α-butylcinnamaldehyde, compound (14) is2-nitro-α-amylcinnamaldehyde and compound (15) is2-nitro-α-hexylcinnamaldehyde. An example reaction scheme for synthesisof 2-nitro-alpha-alkylcinnamaldehyde analogs of the present disclosureis depicted at FIG. 11. Specifically, at FIG. 11, reaction scheme 1100broadly depicts how a 2-nitro-alpha-alkylcinnamaldehyde compound may besynthesized. Reaction scheme 1105 shows a more detailed mechanisticscheme. The synthesis schemes at FIG. 11 may be used, for example, togenerate 2-nitro-alpha-alkylcinnamaldehyde compounds depicted by numeral1110. While not explicitly illustrated at FIGS. 5A-5B, other examples ofstructural analogs of t-CA substituted at both the alpha position andthe ring position may include analogs that include anelectron-withdrawing group at one or more of an ortho, a meta, and apara position of t-CA, and an alkyl group at an alpha position of t-CA.Also relevant to the present disclosure but not included at FIGS. 5A-5Bis hydrocinnamaldehyde and 2-methoxycinnamaldehyde.

Selection of substitution groups and/or positions to substitute t-CA maybe based on increasing lipophilicity as compared to t-CA, for example.In other examples, the selection of substitution groups and/or positionsto substitute t-CA may be based on making the aldehyde section of theparticular analog more reactive with an oxidizing species in CYP2A6, toincrease inhibitor potency. Such selection of substitution groups and/orpositions may be based on a current understanding of how aldehydes areconverted by CYP enzymes to metabolites that form heme adducts. In stillother examples, selection of substitution groups and/or positions may bechosen to stabilize a carbon radical following deformylation, which mayincrease inhibitor potency. For example, adding electron-withdrawinggroups (e.g. F, Cl Br, CF₃ or NO₂) at one or both of the alpha positionand the ring may make the aldehyde more reactive. It may be understoodthat the structural analogs are not limited to those depicted at FIGS.5A-5B, but may include any one of the structures depicted at FIGS.5A-5B, but in a reduced form where the double bond (C═C) (between thealdehyde and the ring) is reduced to a single bond (C—C).

It may be understood that the mechanistic details of how t-CA inhibitsCYP2A6 are not well-established. Thus, it may be understood that thereis no theoretical framework which may readily lead one of ordinary skillin the art to know what structural analogs may lead to inhibitors thatare more potent than t-CA. Moreover, because small structural changes tot-CA may result in differences in the mechanistic details of inhibition,one of ordinary skill may not readily predict which structural analogsof t-CA may increase potency. That is, structural changes may lead tochanges in the reactivity of the molecule, and how it interacts withCYP2A6, and such changes are not readily predicted because the mechanismof action has not been studied extensively.

Based on the above, the following description and examples furtherhighlight the compounds, formulations and methodologies of the presentdisclosure.

I. EVALUATION OF SELECTIVITY, POTENCY AND INHIBITION PARAMETERS OF HUMANCYP2A6 BY T-CA AND STRUCTURAL ANALOGS THEREOF Example 1. ComputationalApproach

An initial approach involved in silico screening using molecularmodeling simulation software (e.g., Autodock and Autodock Vina) wherestructural analogs of t-CA were bound to a crystal structure of CYP2A6(e.g., structure 1Z10) to estimate binding affinity (K_(D)) and bindingorientation between CYP2A6 and each analog. Pymol software may be usedfor rendering images from the docking studies. This initial approach mayprioritize compounds for wet lab experiments. t-CA analogs may thus beprioritized based on the disassociation constants (K_(D)) that thedocking studies provide. K_(D) values indicate affinity, and it may beexpected to identify a structure-affinity relationship for theinteraction of CA-based analogs with CYP2A6. The K_(D) values may becompared with the K_(S) and IC₅₀ values (discussed below) to determinehow well the in silico method predicts affinity values from benchexperiments. FIG. 6 depicts an example dataset 600, showing a partialsummary of the computational docking results to predict affinity (K_(D))of the cinnamaldehyde structural analogs to CYP2A6.

Example 2. IC₅₀ Measurements

Promising candidates (structural analogs of t-CA) for CYP2A6 inhibitionmay be studied by measuring IC₅₀ values, using a recombinant CYP2A6system to measure coumarin hydroxylase activity, which is a marker foractivity of CYP2A6. It may be understood that IC₅₀ values do not provideinformation on the mechanism of inhibition, but rather may be utilizedto provide an early assessment of potency to prioritize compounds formore detailed analysis.

Briefly, potency of t-CA (and structural analogs thereof) was evaluatedby measuring IC₅₀ values in recombinant human CYP2A6 Supersomes™, andhuman liver microsomes. Coumarin hydroxylation, a marker activity forCYP2A6 as discussed above, may be used to measure CYP2A6 activity. A96-well plate assay and fluorescence spectroscopy may be used toquantify the product, 7-hydroxycoumarin.

Referring to Table 1A below, using the methods described above forevaluation of potency, IC₅₀ data is indicated for inhibition of CYP2A6by CA and analogs. Data was fit using GraphPad Prism.

TABLE 1A IC₅₀ data for inhibition of CYP2A6 by cinnamic analogs. Datafit using GraphPad Prism. Cinnamic Analog IC₅₀ (μM)

  Cinnamic aldehyde  6.9 ± 1.2

  Cinnamic alcohol 354.4 ± 4.4

  Cinnamic acid >2500

Based on Table 1A, it can be seen that t-CA is highly selective forinhibiting CYP2A6 as compared to cinnamic alcohol, and cinnamic acid.

Referring to Table 1B, further IC₅₀ data is indicated for select t-CAanalogs of the present disclosure.

TABLE 1B IC₅₀ data for inhibition of CYP2A6 by structural analogs of CAof the present disclosure. Data fit using GraphPad Prism. Inhibitor IC₅₀(uM) 95% CI N α-alkyl substituted analogs α-methyl-CA 5.3 4.4-6.5 9α-amyl-CA 65.0  30.5-138.5 9 α-hexyl-CA 64.7 44.4-94.3 9 α-halogensubstituted analogs α-chloro-CA 3.3 2.8-3.8 9 α-bromo-CA 2.8 2.1-3.8 12Reduced, Oxime, Nitrile, and β-phenyl CA analogs hydro-CA 48.3 40.1-58.19 Cinnamaldehyde 8.0 7.0-9.1 9 oxime cinnamonitrile 8.8  6.7-11.5 9β-phenyl CA 147.6 113.9-191.3 9 ortho-substituted analogs 2-nitro-CA15.1  7.1-23.1 12 2,6-difluoro-CA 3.9 3.0-5.2 9 meta/para-substitutedanalog 3-nitro-CA 409.8 234.7-715.4 8 4-nitro-CA 307.5 175.5-538.9 94-bromo-CA 58.1 51.4-65.6 9 4-chloro-CA 30.1 27.5-33.0 9 4-fluoro-CA30.1 23.8-38.1 9 4-dimethylamino-CA 100.2  88.8-113.2 94-diethylamino-CA 451.4 217.9-935.1 9 4-t-butyl-α-methyl-CA 347.7217.1-557.1 9

Values from the IC₅₀ screens indicate that small substituents (e.g., Cl,Br, and CH₃) at the alpha position (next to the carbonyl group) areallowed and are more potent inhibitors than structural analogs that havelarger substituents (e.g., hexyl and amyl) at the alpha position. Thedata also indicates that replacing the carbonyl group with a cyano (C═N)group or oxime still results in potency similar to cinnamaldehyde. Inregard to substituents on the aromatic ring, the results indicate thatsmall substituents at the ortho position are more potent inhibitors thanmolecules with substituents at the meta and para positions. Also,substitution with electron withdrawing groups (i.e., NO₂, Cl, F, Br) onthe ring is more favorable for inhibition than electron donating groups(e.g., diethylamino). Finally, larger substituents on the ring,especially at the para position are expected to be less potentinhibitors of CYP2A6.

As alluded to above, IC₅₀ data may not predict whether a particularcompound is a time-based inhibitor of CYP2A6. For example, as will bediscussed in further detail below, while α-methyl-cinnamaldehydedisplayed IC₅₀ data similar to that of α-chloro-cinnamaldehyde,α-methyl-cinnamaldehyde is found to not be a time-dependent inhibitor ofCYP2A6, while α-chloro-cinnamaldehyde is found to be a substantiallymore potent time-dependent inhibitor of CYP2A6 than t-CA. Such a findingthus represents an unexpected technical effect that may not be readilypredicted by one of ordinary skill in the art, as will be furtherdiscussed below.

Example 3. Spectral Binding Constant (KS) Measurements

Spectral binding studies were conducted using purified heterologouslyexpressed CYP2A6 in Escherichia coli. Briefly, a solution of CYP2A6 maybe titrated with ligand (CA or structural analog thereof) and monitoredby visible absorption spectroscopy. Absorbance changes (A386-A418) maybe plotted against added ligand concentrations in order to determinedissociation constants.

In more detail, the CYP2A6 enzyme may be heterologously expressed inEscherichia coli and purified. The expression vector, pKK2A6dH may beused to express the enzyme. The vector contains a cDNA sequence thatcodes for human CYP2A6 with a deletion of the N-terminal transmembranesequence, an alteration of several residues at the modified N-terminusto increase expression, and four histidine residues added at theC-terminus to aid in purification. Truncated versions of P450 enzymeshave been shown to retain similar activity as the full-length parentenzymes while allowing for far greater yields of expressed protein andhave been successfully used in kinetic and biophysical studies.Expression from the vector may be induced with the addition of IPTG tobacterial cultures. δ-Aminolevulinic acid is added during expression tosupplement heme biosynthesis. The enzyme may be purified followingpreviously reported protocols. Briefly, following cell disruption, thesolubilized enzyme may be purified using Ni²+-affinity chromatographyand cation-exchange (CM sepharose) chromatography, which may lead toprotein of high purity and P450 content.

Using the above-described methods, spectral ligand binding data wasobtained as depicted at Table 2.

TABLE 2 Spectral ligand binding data to CYP2A6 by t-CA and structuralanalogs of t-CA of the present disclosure. Data fit using GraphPadPrism. Inhibitor K_(s) (uM) t-cinnamaldehyde 14.9o-methoxycinnamaldehyde 1.6 hydrocinnamaldehyde 44.8 α-methyl-CA 6.8α-ethyl-CA 3.0 α-propyl-CA 7.8 α-isopropyl-CA 3.2 α-butyl-CA 36.8α-chloro-CA 9.6 α-bromo-CA 8.2 o-nitrocinnamaldehyde 47.52,6-difluoro-CA 7.8 4-chloro-CA 48.9For illustrative purposes, Table 2 depicts a select number of structuralanalogs of the present disclosure. Again, α-methyl-cinnamaldehyde, forexample, displays binding affinity for CYP2A6 that is on par withα-chloro-cinnamaldehyde, yet α-methyl-cinnamaldehyde is determined (seebelow) to not be a time-based inhibitor of CYP2A6, whereasα-chloro-cinnamaldehyde is found to be a substantially more potenttime-dependent inhibitor of CYP2A6 than t-CA.

Example 4. Evaluation of Structural Analogs for Time DependentInhibition of CPY2A6

Mechanism-based inhibitors are most easily identified by inhibition thatincreases with time. Since this type of inhibition also requires thatthe inhibitor is initially metabolized by the target enzyme, MBIs may beidentified by evaluating the inhibition in the presence and absence ofcofactors that are required for activity, such as NADPH for CYPs. Ourstudies indicate that t-CA inhibition (and structural analogs thereof)of CYP2A6 increases with time and is profoundly greater when t-CA (andstructural analogs thereof), NADPH, and CYP2A6 are incubated together ascompared to samples without NADPH. FIG. 7A depicts a representativeexample of the type of data illustrative of mechanism-based inhibitionusing t-CA as the inhibitor, where the assay is for time-dependentinhibition of 7-hydroxycoumarin formation. The data depicted at FIG. 7Awas generated with recombinant CYP2A6, but it may be understood thatsuch data may also be obtained similarly using CYP2A6 Supersomes™ andhuman liver microsomes.

By obtaining the type of data as that depicted at FIG. 7A, plots such asthat depicted at FIG. 7B (a representative example using2-nitro-α-methylcinnamaldehyde) may be obtained and K_(inact) and K_(I)values may be obtained using nonlinear regression (e.g., via GraphpadPrism) and equation 1:K _(obs) =k _(inact)*[I]/K _(I)+[I]  (1)

Table 3 illustrates a summary of time-dependent inhibition parameters ofCYP2A6 for t-CA and structural analogs of the present disclosure usingcoumarin hydroxylase activity as a readout of inhibition in arecombinant CYP2A6 system. As indicated, α-chloro-cinnamaldehyde,o-nitro-CA (2-nitro-CA), o-nitro-α-methyl-CA (2-nitro-α-methyl-CA),o-trifluoromethyl-CA (2-trifluoromethyl-CA) and 2,6-difluoromethyl-CAare all more potent inhibitors than t-CA, with α-chloro-cinnamaldehydebeing over 30-fold more potent. Such a result is unexpected and cannotbe readily predicted by IC₅₀ data and binding data as discussed above.Furthermore, the substantial increased potency observed forα-chloro-cinnamaldehyde cannot be generalized as being simply due to thepresence of a halogen (e.g., electron-withdrawing group), as α-bromo-CAshowed less potency as compared to t-CA, and α-fluoro-CA (refer to FIG.8) was shown to not be a time-dependent inhibitor at all. Thus, thepotency observed for α-chloro-cinnamaldehyde represents an unexpectedresult. For example, based on the result for α-fluoro-CA (refer to FIG.8) that shows α-fluoro-CA is not a time-dependent inhibitor, it may beexpected that α-chloro-cinnamaldehyde would also not be a time-dependentinhibitor, but that is not what is observed.

TABLE 3 Time-dependent inhibition screen of t-CA and structural analogsthereof using a recombinant CYP2A6 system Fold difference k_(inact)k_(I) (with respect Analog (min⁻¹) (μM) k_(inact)/k_(I) to CA) t-CA0.039 27.2 0.00143 1 o-methoxy-CA not measured not measured n/a n/aHydro-CA 0.116 85 0.00136 0.96 α-bromo-CA 0.017 36.3 0.00047 0.32α-chloro-CA 0.15 3.4 0.044 30.7 o-nitro-CA 0.223 13.9 0.0160 11.2o-nitro-α-methyl-CA 0.243 19.6 0.0124 8.7 m-nitro-CA 0.083 89.6 0.000930.65 p-nitro-CA 0.039 92.1 0.00042 0.29 o-trifluoromethyl-CA 0.142 52.60.00270 1.9 2,6-difluoromethyl-CA 0.244 47.2 0.00517 3.6

In fact, a number of structural analogs not depicted at Table 3 abovewere found to not be time-dependent inhibitors of CYP2A6. As mentionedabove, examples of such compounds are depicted at FIG. 8. Morespecifically, the compounds depicted at FIG. 8 displayed similarprofiles as that shown above at FIG. 7A for NADPH only (diamonds at FIG.7A), or t-CA only (black squares at FIG. 7A), as compared totime-dependent inhibitors (circles at FIG. 7A). Compounds found not tobe time-dependent inhibitors included 2-hydroxy-t-CA, hydro-CA,α-methyl-CA, 2-methyl-CA, α-fluoro-CA, α-ethyl-CA, α-pentyl-CA,α-butyl-CA, α-isopropyl-CA, α-propyl-CA α-butyl-CA, and α-hexyl-CA.

The fact that t-CA structural analogs substituted at the alpha positionwith alkyl groups (e.g., α-methyl-CA, 2-methyl-CA, α-ethyl-CA,α-pentyl-CA, α-butyl-CA, α-isopropyl-CA, α-propyl-CA α-butyl-CA, andα-hexyl-CA) were found to not be time-dependent inhibitors renders thefinding that o-nitro-α-methyl-CA shows improved inhibition parameters(8.7-fold increase over t-CA) unexpected.

The above-discussed approach regarding evaluation of selectivity,potency and inhibition parameters of human CYP2A6 by t-CA and structuralanalogs thereof may be similarly applied to evaluation of the sameparameters with regard to CYP2A13. As one representative example,inhibition of CYP2A13 as monitored via coumarin hydroxylase activity asa readout of inhibition in a recombinant CYP2A13 system shows that2-nitro-cinnamaldehyde has a K_(I) of 30.95 μM, and a K_(inact) of0.12161 min⁻¹, resulting in a K_(inact)/K_(I) of 0.00407.

II. EVALUATION OF ‘DRUGGABILITY’ OF CA AND STRUCTURAL ANALOGS THEREOF ASTOBACCO CESSATION AGENTS

There are many reasons why a potent in vitro inhibitor may not become aneffective drug, including instability, toxicity, and drug-druginteractions. Thus, t-CA and structural analogs thereof may be evaluatedas to promising drug-like characteristics, discussed in the examplesbelow.

Example 5. Evaluation of the Potential of the Interaction between t-CA(And Structural Analogs Thereof) and CYP2A6-Mediated Nicotine Metabolism

To evaluate the potential of the interaction between t-CA (andstructural analogs thereof) and CYP2A6-mediated nicotine metabolism, amodel may be used that incorporates mechanism-based inhibitionparameters (i.e., K_(I) and k_(inact)), an intrinsic degradation rate ofCYP2A6 in the absence of inhibitors (k_(deg)), and a fraction of anicotine dose metabolized by CYP2A6 (f_(m)), to predict changes in thenicotine area under the curve (AUC), a parameter indicative of apatient's exposure to a drug over time. Equation 2 shows how the AUCchanges in the presence of an inhibitor. In the context of this example,AUCi/AUC refers to the AUC for nicotine in the presence of CA divided bythe AUC in the absence of t-CA.

$\begin{matrix}{{{Prediction}\mspace{14mu}{of}\mspace{14mu} A\; U\; C\mspace{14mu}{changes}\mspace{14mu}{in}\mspace{14mu}{the}\mspace{14mu}{presence}\mspace{14mu}{of}\mspace{14mu} a\mspace{14mu}{mechanism}\text{-}{based}\mspace{14mu}{{inhibitor}.\text{}\mspace{20mu}\frac{{AUC}_{i}}{AUC}}} = \frac{1}{( \frac{f_{m,{CYP}}}{1 + ( \frac{k_{inact} \cdot \lbrack I\rbrack}{k_{\deg} \cdot ( {K_{I} + \lbrack I\rbrack} )} )} ) + ( {1 - f_{m,{CYP}}} )}} & {{Equation}\mspace{14mu} 2}\end{matrix}$

Using data from our initial studies, Equation 2 predicts profoundchanges in nicotine AUC, even at low plasma concentrations of t-CA(depicted as a representative example at Table 4). That is, a 4.5-foldchange in nicotine AUC is predicted at just 1 μM t-CA.

TABLE 4 Predicted changes in AUC for oral nicotine in the presence ofvarying concentrations of CA. AUC Fold AUC Fold [CA] k_(deg) ^(e)Increase Increase μM f_(m) ^(d) (per min) Supersomes Microsomes 0.1 .86.00044 1.4 1.8 1 .86 .00044 3.4 4.5 10 .86 .00044 6.0 6.4 CA = cinnamicaldehyde; ^(d)f_(m) = fraction of a nicotine dose metabolized by CYP2A6;^(e)k_(deg) = degradation rate of CYP2A6 in vivo.

In humans, t-CA is completely absorbed upon oral administration. Thishas also observed in rats, for which full pharmacokinetic studies areavailable. Converting the dose in rats to humanized equivalent doses canestimate the doses necessary to achieve low μM concentrations of CA inhumans. The Food and Drug Administration suggests using the body surfacearea (BSA) normalization method to extrapolate animal doses to humandoses. In rats a single 250 mg/kg oral dose generated a t-CA bloodconcentration of 7.6 μM. With the BSA method, t-CA doses of 32 mg and320 mg per day are estimated to achieve concentrations of 0.1 and 1 μMin humans. Notably, the estimated range of t-CA exposure when cinnamonpowder is used for diabetes is 8 to 275 mg/day.

Since smokers titrate nicotine plasma concentrations to a defined range,and poor metabolizers smoke fewer cigarettes than rapid metabolizers,the data presented in Example 5 suggests that t-CA can profoundly impactnicotine clearance, carcinogen exposure, and potentially impact dosingof nicotine replacement therapy. Substantial and prolonged inactivationmay occur during first-pass liver exposure in which the concentration oft-CA may be higher than the concentration in the systemic circulation.Therefore, a dose that results in 1 μM t-CA (or structural analogsthereof) in the blood, may result in even greater reduction in nicotinemetabolism than predicted by the model, especially for individuals whoare rapid metabolizers of nicotine. Overall the modeling andpharmacogenetic data support the hypothesis that t-CA (and structuralanalogs thereof) may impact smoking cessation rates.

Turning to Table 5, it depicts exemplary K_(I), k_(inact), and AUCpredictions for several structural analogs of the present disclosure, ascompared to t-CA.

TABLE 5 k_(I), k_(inact), and AUC predictions k_(inact) k_(I) AUC foldAnalog (min⁻¹) (uM) k_(inact)/k_(I) increase t-CA 0.056 27.2 0.0020591.71-2.69 α-chloro-CA .15 3.4 0.043989 2.44-4.22 α-bromo-CA .017 36.30.000463 1.35-1.84 hydro-CA .116 85.0 0.001365 1.71-2.67

To empirically determine inhibition of nicotine metabolism, anHPLC-MS/MS method may comprise use of a Phenomenex Kinetix C18 column(2.1×50 mm; 2.6 micron) and a mobile phase consisting of 0.2% ammonia inwater (solvent A) and acetonitrile (solvent B). To separate nicotine,cotinine, and the internal standard (D3-cotinine) the mobile phase maybe ramped from 5%-95% B over 5 minutes, followed by a column wash byholding 95% B for 1 minute and re-equilibrating to 5% B for 3 minutes.Cotinine and Nicotine may be quantified by multiple reaction monitoring(MRM) using the 177.4/80.2 Da (parent/transition) fragment for cotinine,the 163.1/117.2 fragment for nicotine and the 180.06/100.9 fragment forD3-cotinine. To determine time dependent inhibition parameters humanliver microsomes (200-donor pool; 5 mg/ml) may be preincubated in atotal volume of 140 μl for 5 minutes at 37° C. in buffer (50 mMpotassium phosphate, pH=7.4). Aliquots (20-μl) may be removed from theprimary incubation at multiple time points and diluted to 200 μl inincubation buffer containing cytosol (1 mg/ml protein), nicotine (50 μM)and NADPH (1 mM), and incubated for an additional 15 minutes at 37° C.Incubations may be terminated with 200 μl acetonitrile and 100 μl of 5μg/mL D3-cotinine (internal standard) may be added. The samples may beanalyzed by the HPLC-MS/MS method described above. The inhibitorsidentified to be potent inhibitors of CYP2A6 may be assessed in humanhepatocytes, a more physiologically relevant system.

Example 6. Evaluation of Metabolic Stability and Drug-Likeness of T-CAand Structural Analogs Thereof

To evaluate metabolic stability and drug-likeness of t-CA and structuralanalogs thereof, experiments may be conducted in human liver microsomeswith cytosol. t-CA and structural analogs thereof may be incubated inmicrosomes (0.5-0.75 mg/mL) and cytosol with phosphate buffer (pH=7.4)and NADPH for 60 minutes, where aliquots may be analyzed by HPLC atspecific time points. Standard curves may be used to quantify loss ofeach of t-CA and structural analogs thereof, as a function of time.Specifically, an HPLC-fluorescence assay for CA may be used to ascertainstability, and which may similarly be used to monitor stability ofstructural analogs.

Lipinkski's rule of five (RO5) was used to evaluate the drug-likeness ofCA and structural analogs thereof. Each analog may be given a scorebetween 0 (not drug-like) and 4 (drug-like) based on how well it fitsthe criteria of the RO5, the RO5 comprising 1) molecular weight lessthan 500 Da, log P<5 (measure of lipophilicity), number of hydrogen bonddonors (HBD)<5, and the number of hydrogen bond acceptors (HBA)<10.Molecular weight and log P values may be obtained from PubChem andChemSpider. A representative sample is depicted at Table 6.

TABLE 6 Drug-likeness of CA and CA analogs Structure MW Log P HBD HBAScore

132.16 1.9 0 1 4

211.06 2.7 0 1 4

168.14 2.3 0 1 4

III. DEVELOPMENT AND EVALUATION OF FORMULATIONS OF T-CA (AND STRUCTURALANALOGS THEREOF) FOR BIOAVAILABILITY AND INHIBITION OF NICOTINEMETABOLISM

As discussed above, previous results indicate that t-CA (and structuralanalogs thereof) is a MBI of CYP2A6, predicting substantial changes innicotine clearance. A potential barrier for t-CA (and structural analogsthereof) to serve as a tobacco cessation agent may be rapid degradationor poor adsorption in the gastrointestinal tract or the liver. Studiesin rats have shown that t-CA is metabolized rather quickly, suggestingthat dosages may need to be relatively high to achieve sufficient CYP2A6inhibition in vivo. A principal of toxicology is the relationshipbetween dose and toxicity, with high-dosed drugs typically associatedwith a greater risk for toxicity. To overcome such issues, formulationsand prodrug forms (oxime-ethers) of t-CA (and structural analogsthereof) are discussed herein. Both may improve absorption,distribution, metabolism, and excretion (ADME) properties and allow forlower dosing while maintaining effectiveness. Thus, an aspect of thepresent disclosure is evaluation of formulations of t-CA (and structuralanalogs thereof) for loading, stability, and t-CA (and structuralanalogs thereof) release characteristics (see III.i), synthesis ofoxime-ether prodrug forms of t-CA (and structural analogs thereof) aswell as evaluation of their metabolic stability (see III.ii),measurement of intestinal absorption and CYP2A6 inhibition potency (seeIII.iii), and use of a rat model to determine systemic exposure of CAand to measure the PK parameters of the oxime-ether prodrug (seeIII.iv). Turning to FIG. 9A, an example scheme 900 depicts how t-CA (orstructural analogs thereof) may be released from an oxime-ether by oneof two routes (route I or route II which includes steps II.a and II.b).The oxime-ether may be hydrolyzed in the presence of water (route I), ormay first undergo O-dealkylation by cytochrome P450 enzymes in the liver(route II.a), followed by hydrolysis (route II.b). Turning to FIG. 9B,example illustration 950 depicts oxime ethers of the present disclosure.The oxime ethers depicted at FIG. 9B are shown with regard to t-CA, butit may be understood that t-CA as depicted at FIG. 9B may be replacedwith any of the structural analogs depicted at FIGS. 5A-5B.

III.i Evaluation of Formulations of CA for Loading, Stability and CA(and Structural Analogs Thereof) Release

Two formulations of CA (and structural analogs thereof) may be prepared,a self-emulsifying drug delivery system (SEDDS), and a co-solventsolution using Pluronic® polymers or polaxamers which are tri-blockcopolymers of poly(ethylene oxide)-poly(propylene oxide)-poly(ethyleneoxide) (PEO-PPO-PEO). The SEDDS system may be developed by determiningthe best surfactant/mixture of surfactants that will match theHydrophile-Lipophile Balance (HLB) value for the t-CA (or structuralanalogs thereof) oil. The system may be assessed for t-CA (or structuralanalogs thereof) loading and stability at room temperature by monitoringsize and zeta potential. t-CA (and structural analogs thereof) releasemay be assessed by a dialysis method under sink conditions. The SEDDSmay deliver nanoscale delivery of t-CA (and structural analogs thereof)at high concentrations to allow for greater absorption similar to themarketed SEDDS formulation, Neoral®.

The co-solvent system may be prepared by determining the best polaxamersto enhance the oral bioavailability of the CA. Polaxamers are tri-blockco-polymers and biologic response modifiers that have shown the abilityto increase the bioavailability and alter the PK of moleculesco-delivered with them. Thus CA incorporated into polaxamers may beprepared by solvation method. One potential issue includes challengesassociated with solubilizing high concentrations of CA (and structuralanalogs thereof) in the co-solvent system using the polaxamers. Toaddress this, other block co-polymers like PEG-PCL or PEG-PLA, whichhave different polarities, may be utilized to achieve higher drugconcentrations.

III.ii Synthesis of Prodrug Forms of CA Based on an Oxime-Ether ChemicalScaffold and Evaluation of Metabolic Stability

Synthesis of oxime compounds was conducted via condensation of t-CA (andstructural analogs thereof) with a variety of hydroxylamine ethers(H₂N—OR), where R is one of methyl, ethyl, etc. The hydroxylaminederivatives are available from commercial sources and FIG. 10 broadlydepicts reaction scheme 1000 for synthesizing the oxime compounds whichmay include heating t-CA and H₂N—OR together in a protic solvent.Reaction conditions may be optimized to suit the reactivity ofparticular combinations. A weak acid catalyst at sub-stoichiometricquantities may be utilized to accelerate the reaction. Metabolicstability and generation of t-CA (and structural analogs thereof) may beassessed using human liver microsomes with cytosol. The oxime-ethers maybe incubated in microsomes and cytosol with phosphate buffer. Aliquotsmay be analyzed by LC-MS/MS at specific time points to evaluate adegradation rate of the prodrug as a function of time. Standard curvesof t-CA (and structural analogs thereof) may be generated to quantify arate of generation of t-CA (and structural analogs thereof).

III.iii Assessment of Intestinal Absorption of the Above-MentionedFormulations and the Prodrug Form of CA (and Structural Analogs Thereof)Assessed Using CACO-2 Cells, and CYP2A6 Inhibition Potency Assessed inHuman Hepatocytes

Delivery systems (the above-mentioned formulations) may be assessed invitro in Caco-2 models to determine efflux across the monolayer. Caco-2cells may be grown to confluence over 20 days in 24 well transwellplates, and t-CA alone (or structural analogs thereof), in formulations,or as the oxime prodrug may be added to the apical insert at variousconcentrations. The wells may be incubated at 37° C. with shaking. At 0,60, and 120 min 20 μL samples may be withdrawn from the apical side, and200 μL samples may be taken from the basolateral side at 0, 15, 30, 45,60, 90, and 120 min. t-CA (and structural analogs thereof)concentrations may be assessed by LC-MS/MS, and the apparent partitioncoefficient (P_(app)) may be calculated. Inhibition of nicotinemetabolism may be evaluated in human liver microsomes with cytosol,followed by studies in human hepatocytes (a more physiologicallyrelevant system). Oxime flux may additionally be determined across theCaco-2 monolayer for comparison to t-CA (and structural analogsthereof).

III.iv Measurement of In Vivo PK Parameters of an Oxime-Ether Prodrug toDetermine a Resulting Systemic Exposure of t-CA from theFormulations/Prodrug

Adult male Sprague-Dawley rats (300-400 g) may be cannulated. Animalsmay be divided into 3 groups of 12 animals: one for each optimized t-CA(and structural analogs thereof) formulation, and oxime prodrug (togenerate t-CA or structural analogs thereof). Animals may be dosedorally via gavage with t-CA (or structural analogs thereof) in theformulations at 250 mg/kg; a dose below the reported LD₅₀. At 5, 15, 30,60, 120, 180, 360, 720, and 1440 minutes post-administration 0.1-0.2 mLof blood may be collected. t-CA (and structural analogs thereof)concentrations may be assessed using LC-MS/MS, and PK parameters may bedetermined using WinNonlin. PK data may be incorporated into PBPKmodels.

Higher AUC values for the t-CA (and structural analogs thereof) informulations and oxime-ether prodrug may be expected. The formulationsmay deliver higher doses of CA (and structural analogs thereof) in boththe SEDDS and the co-solvent system, as in both systems, the presence ofamphipathic molecules may enhance the absorption of various moleculesorally.

In this way, individuals with a dependence on nicotine may be treated toreduce said dependence. Reducing said dependence may in turn reduce apotential for adverse health implications (e.g., lower a risk of cancer,where the cancer is one or more of lung cancer, prostate cancer, bladdercancer, pancreatic cancer, head and neck cancers, esophageal cancer,kidney cancer, stomach cancer, colon cancer, acute myelogenous leukemia(AML), ovarian cancer, breast cancer, liver cancer, cervical cancer, andskin cancer) stemming from the use of nicotine-containing products.Thus, in some examples the compounds, compositions and methods of usediscussed herein may relate to reducing a risk of cancer in a subjectwith a nicotine dependence.

In another embodiment, a pharmaceutical composition of the presentdisclosure may comprise one or more structural analog(s) (refer to FIGS.5A-5B) of the present disclosure, where the structural analog is amechanism-based inhibitor of CYP2A6. In one example, the composition mayfurther comprise one or more of an oil and a surfactant (or one or moredifferent oils and one or more different surfactants). In some examples,the composition may be capable of self-emulsifying in agastro-intestinal environment, or other aqueous environment. In someexamples, the composition may comprise less than 10%, less than 5%, orless than 1% by weight water. In some examples, the composition mayinclude one or more co-solvents. The one or more co-solvents maycomprise polyethylene glycol 300, polyethylene glycol 400, and propyleneglycol, for example. In some examples, the oil may comprise a singlelong chain triglyceride, a single medium chain triglyceride, a mediumchain monoglyceride, and a medium chain diglyceride. In some examples,the oil may be a blend of a monoglyceride or a diglyceride blended witheither a long chain triglyceride or a medium chain triglyceride. In someexamples the oil may be castor oil. In some examples, the surfactant maybe polysorbate 80. In some examples, the structural analog(s) may beencapsulated in an encapsulating nanosphere. In some examples, theencapsulating nanosphere may be formed from one or more tri-blockcopolymers. In some examples, the one or more structural analog(s) maybe incorporated into the encapsulating nanosphere via a solvationmethod. In some examples, the tri-block copolymers may be selected inorder to enhance an oral bioavailability of the one or more structuralanalog(s). In some examples, the tri-block copolymers may comprise PEGPCL and/or PEG PLA.

The following claims particularly point out certain combinations andsub-combinations regarded as novel and non-obvious. These claims mayrefer to “an” element or “a first” element or the equivalent thereof.Such claims should be understood to include incorporation of one or moresuch elements, neither requiring nor excluding two or more suchelements. Other combinations and sub-combinations of the disclosedfeatures, functions, elements, and/or properties may be claimed throughamendment of the present claims or through presentation of new claims inthis or a related application. Such claims, whether broader, narrower,equal, or different in scope to the original claims, also are regardedas included within the subject matter of the present disclosure.

The invention claimed is:
 1. A method for treating an individual with anaddiction to nicotine, comprising administering to the individual acompound of one of the following chemical structure:

wherein the compound is one of 2-nitro-α-methylcinnamaldehyde,2-nitro-α-ethylcinnamaldehyde, 2-nitro-α-isopropylcinnamaldehyde,2-nitro-α-propylcinnamaldehyde, 2-nitro-α-butylcinnamaldehyde,2-nitro-α-amylcinnamaldehyde and 2-nitro-α-hexylcinnamaldehyde, andwherein a rate at which nicotine is metabolized is reduced uponadministration of the compound to the individual.
 2. The method of claim1, wherein the compound is administered orally to the individual.
 3. Themethod of claim 1, further comprising selecting the compound based onwhether the individual is an ultrafast, fast, intermediate, or slowmetabolizer of nicotine.